Ligand-grafted nanoparticles as tools to study L-type calcium channel and β-adrenergic receptor function on surface and T-tubule membranes

The cell membrane of adult ventricular myocytes (AVMs) is characterized by many invaginations, constituting the transverse tubule (TT) system. TT membrane (TTM) is continuous with the outer surface membrane (OSM) to allow the rapid electrical coupling during the action potential and represents about 30% of the total cell membrane. Most membrane proteins, such as L-type Ca2+ channels (LTCC) and β-adrenergic receptors (β- AR) are located in both OSM and TTM, but at different densities. However, how the location of these proteins in OSM vs. TTM impacts on their function remains poorly understood, mainly because of the impossibility to selectively activate or inhibit a protein in one compartment without acting on its counterpart in the other compartment. For example, an important function of the TT system is to provide proximity between LTCCs in TTM and ryanodine receptor type 2 in the sarcoplasmic reticulum membrane. However, the role of their counterparts located in OSM is unknown. The goal of our study is to meet this challenge using an innovative nanotechnology approach. We designed nanoparticles (NPs) with immobilized ligands on their surface in order to prevent their access to TTM. The first step was to graft ligands on 5kDa PEG (polyethylene glycol) to favour ligand flexibility. PEG- and NPs-ligand affinity were determined by radioligand binding assays. PEG- and NPs-ligand potency were evaluated by FRET experiments. Confocal microscopy has been used to visualize the membrane distribution of the fluorescent PEGs and NPs after incubation of AVMs. PEG-ligand affinity and potency are lower than affinity and potency of free ligand. The confocal microscopy assay shows that neither NPs nor PEGs penetrate in the TT network. PEG-ligands seem to act on proteins located on the surface membrane. After this validation, functional experiments will be realized to discriminate the respective role of β-AR and LTCC subpopulations.